Abstract: The current antimicrobial resistance crisis is caused by the rise and spread of drug resistant pathogens. According to the WHO, drug-resistant E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, S. aureus and M. tuberculosis are of especial concern. We are now facing pathogens that are resistant to most, or all, currently available antibiotics. Slowing down of discovery and a very modest pipeline of novel compounds are responsible for the crisis. The last novel compound active against Gram-positive pathogens introduced to the clinic, daptomycin, was discovered over 30 years ago; the last class of compounds acting against Gram negative bacteria, the fluoroquinolones, was developed in the 60s. We have used a number of approaches to exploit an untapped source of antimicrobials ? uncultured bacteria that make up the majority of species of microorganisms. Cultivation in situ led to the discovery of >30 new compounds. One of these, teixobactin, represents a novel class of cell wall acting antibiotics and is in IND-enabling studies against MRSA Staphylococcus aureus. Teixobactin has a unique property, there is no detectable resistance to this compound. Teixobactin binds two immutable targets, lipid II, precursors of peptidoglycan, and lipid III, precursor of wall teichoic acid, accounting for lack of resistance (Ling et al., 2015). Novo29 is another novel compound that binds the same targets. Interestingly, both antibiotics are produced by members of a new genus, Eleftheria, a Gram-negative ?- proteobacteria. Given their ability to produce unusual compounds, we assembled an additional set of Eleftheria isolates from our collection and screened them for antimicrobials, but did not initially observe activity. Whole genome sequencing shows that Eleftheria contain PKS and NRPS operons for the production of secondary metabolites that appear to be silent. We then used media spiked with different amino acids to induce expression, and this led to the production of several antimicrobials that we are currently characterizing. Our preliminary findings suggest that Eleftheria is an attractive genus of bacteria to search for novel antibiotics. The goal of this Phase I project is to examine the potential of this genus to produce antibiotics and identify a novel lead compound for further development. We identified the antibiotic resistance profile of Eleftheria, and will use this to selectively isolate additional members of this group. Spiking of amino acids, as well as mutagenesis will be used to turn on silent operons. The search will be guided by whole genome analysis of isolates, which will indicate the presence of biosynthetic gene clusters. We will focus on isolates with BGCs that lack close homologs. Bioassay-guided fractionation will lead to purification of compounds. Spectrum of activity will be determined, and we will give preference to compounds acting against Gram negative bacteria. Compounds with minimal cytotoxicity will be advanced to mode of action studies. In vivo efficacy will serve as the endpoint of this project. The goal is to isolate a least 1 novel lead compound with efficacy in a mouse model, ready for subsequent drug development.